The goals of this application are to understand the control of the coagulation mechanism that occurs through the regulation of factor VIII (fVIII) function and the mechanisms underlying pathogenic anti-fVIII Abs. The regulatory processes addressed in this project include the activation of the fVIII-von Willebrand factor (vWf) complex and the control of intrinsic pathway activator function, including the decay of activated fVIII (fVIIIa) activity that occurs through A2 subunit dissociation. The role of the fVIII C2 domain in the activation of fVIII and the mechanism of inhibition of fVIII by anti-C2 antibodies (Abs) will be determined. A large Ab epitope map of the C2 domain has been developed. This has led to the identification of a novel class of C2 domain Abs that inhibit the activation of fVIII by either thrombin or factor Xa. These Abs make a dominant contribution to the inhibitory immune response to fVIII in hemophilia A mice and in humans. Additionally, they produce a bleeding diathesis in mice. The inhibition of fVIII activation by these Abs is surprising because their epitopes are remote from proteolytic cleavage sites that are involved in the activation of fVIII. Experiments will be done to determine which, if any, proteolytic cleavages in fVIII catalyzed by thrombin and factor Xa are inhibited by these Abs. Additionally, the mechanism of inhibition by the Abs will be investigated. The kinetics of binding of anti-C2 Abs to fVIII in the presence and absence of vWf also will be analyzed. This will allow testing of the hypothesis that the behavior of fVIII inhibitors is governed by the so-called affinity limit of Abs for their antigens. Additionally, these studies are directed toward understanding the complex behavior of many inhibitors, which can include partial inhibition of fVIII and complex, non-second order kinetics. Additionally, high-resolution mapping of anti-C2 epitopes by site directed mutagenesis will be performed. This proposal has potential clinical implications, including the development of novel recombinant fVIII molecules with improved hemostatic efficacy in the treatment of patients with hemophilia A and improved diagnostics for fVIII inhibitor patients.